The mitotic spindle is a bipolar, self-organizing protein machine that uses multiple microtubule (MT)-based motor proteins to assemble itself and to segregate sister chromatids. The broad aim of the work proposed here is to understand the roles of these motors in the mechanism of mitosis. The conceptual framework underlying the proposal is that spindle morphogenesis, characterized by changes in the positioning of spindle poles, involves transitions between steady state structures. Each steady state structure depends on a delicate balance of forces generated by multiple antagonistic and complementary motors, and tipping this balance by up- or down-regulating subsets of motors drives transitions between steady states. These transitions equate to specific mitotic movements, such as the repositioning of spindle poles. The specific aims are designed to test elements of this multiple motor-dependent transient steady state hypothesis and to refine our current model that describes the role of bipolar kinesin motors, C-terminal kinesin motors and cytoplasmic dynein in spindle pole positioning. The aims are 1. To test the roles of additional mitotic motors in pole positioning, 2. To test the hypothesis that down-regulating the C-terminal kinesin initiates anaphase spindle elongation, 3. To use structure-function assays in vivo and in vitro to test the hypothesis that bipolar kinesins and the C-terminal kinesins use a sliding filament mechanism to position spindle poles 4. To test the roles of motor-driven MT sliding versus MT assembly dynamics in pole positioning, and 5. To develop quantitative models that explain the motor-dependent spindle pole separation curves. Our research strategy will exploit the Drosophila syncytial blastoderm-stage embryo, which is amenable to biochemical, genetic and cytological analysis of mitotic motor function. The results will contribute to an understanding of the basic mechanisms of mitosis and may provide insights into some of the dysfunctions in spindle action and chromosome segregation that characterize certain birth de